Site-specific recombination is one of the means by which the arrangement of genes is altered in nature. The AIDS virus uses it to incorporate its genome into the chromosomes of immune cells. Immunoglobulin genes rearrange by site-specific recombination iii order to create the wide variety of antibodies available to combat disease. The study of site-specific recombination also provides a model for investigating the fundamental properties of complex protein-nucleic acid interactions. The proposed experiments will study site-specific recombination as exemplified by the integration of bacteriophage lambda DNA into a site in the Escherichia coli chromosome. The lambda reaction is believed to proceed as follows: A complex containing the phage protein Int and the E. coli protein integration host factor (IHF) organizes at a DNA site, attP, on the phage chromosome. This structure, the intasome, then binds the bacterial integration site, attB. The resultant synapsed intermediate undergoes the DNA strand breakages, rearrangements, and ligations needed for integration. This work seeks to dissect the interaction between the intasome and attB. This is rather weak and requires special techniques for measurement. The kinetic properties of the recombination reaction can provide the desired information. The amount of saturation of the reaction rate as a function of the attB concentration will reveal the steady state dissociation constant, K-Int, of the intasome and attB . Quantitation of recombination at high attB concentrations clearly indicates evidence for saturation above 200 nM attB. K-Int will be corroborated by measuring inhibition of recombination by variant attBs that have altered sequences which are recognized by the intasome but unable to complete recombination. The inhibition constant is expected to be similar to the dissociation constant. The determined in vitro parameters will then be compared to the in vivo parameters. These will be estimated by quantitating phage integration into a plasmid in E. coli in the presence of competing nonfunctional attB sites. It will then be determined whether K-Int reflects only the protein recognition of the attB sequence or whether it also reveals information about the strand exchanges and rearrangements. This will be done by comparing the inhibition of a set of modified attBs whose sequence blocks their ability to recombine at specific points along the pathway. The kinetic experiments also allow the determination of the maximal rate of strand exchange. By studying how this rate is perturbed by factors such as temperature and sequence, the nature of the exchange will be discerned.